The calcium-sensitive photoprotein aequorin and the green fluorescent protein were isolated and purified from Aequorea forskålea. Purified aequorin shows electrophoretic microheterogeneity but appears as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequence determination by the automated Edman technique revealed a single NH2-terminal sequence: Val-Lys-Leu-(Thr)-Pro-Asp- Phe - Asn- Asn - Pro-(¯)- Trp- Ile-Gly-Arg-His-aequorin exists as a single polypeptide chain. Apparent molecular weights were determined by sedimentation equilibrium, urea-sodium dodecyl sulfate/polyacrylamide gel electrophoresis, gel filtration of native protein, and gel filtration of denatured protein in 6 M guanidine hydrochloride; all methods suggest an apparent molecular weight of 19 500 ±2000 for aequorin. Under some conditions the protein di-merizes by disulfide bond formation. Aequorin has a sedimentation coefficient of 2.31 S and a Stokes radius of ~19 Å; the extinction coefficient (E1cm1%) was calculated to be 27.1. Amino acid analysis revealed a slight preponderance of acidic residues; no carbohydrate moieties were found. Aequorin contains at least one free sulfhydryl group, chemical modification of which results in irreversible loss of luminescent activity. The apparent molecular weight of the green fluorescent protein is 30 000 when determined by urea-sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gel filtration of denatured protein in 6 M guanidine hydrochloride. Amino acid analysis revealed an absence of tryptophan.
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